CN115322391B - Two-dimensional Cu-CAT nanosheets with enzyme-like activity and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of new nanometer materials, and in particular relates to a two-dimensional Cu-CAT nanosheet with enzyme-like activity and a preparation method and application thereof. Provided is a two-dimensional Cu‑CAT nanosheet with enzyme-like activity. The nanosheet is a two-dimensional (2D) nanomaterial, which has the advantages of many active sites, high sensitivity, and good specificity, and is conducive to the generation of colorimetric signals. ; At the same time, it also provides the application of the nanosheet, which is a colorimetric biosensor for rapid detection of AChE and its inhibitors, which has good detection selectivity for AChE and low detection limit; meanwhile, it can detect or screen AChE inhibitors.

Description

A two-dimensional Cu-CAT nanosheet with enzyme-like activity and its preparation method and application

technical field

The invention belongs to the technical field of new nanometer materials, and in particular relates to a two-dimensional Cu-CAT nanosheet with enzyme-like activity, a preparation method and application thereof.

Background technique

Alzheimer's disease is a common chronic neurodegenerative disease characterized by the progressive decline of memory and learning ability, and its pathogenesis is generally considered to be caused by the production and accumulation of amyloid-β peptide (aβ) in the brain of. Acetylcholinesterase (AChE) is one of the most important enzymes in the mammalian central nervous system. In the presence of AChE, Aβ peptides can be deposited to form amyloid plaques and neurofibrillary tangles. It is generally believed that the abnormal expression of AChE is highly related to Alzheimer's disease, and inhibiting the activity of AChE can effectively control the progress of Alzheimer's disease. Therefore, AChE is considered to be one of the most important biomarkers for Alzheimer's disease. Establishing a simple and sensitive AChE detection method is of great significance for early diagnosis and evaluation of Alzheimer's disease progression. In addition, since neostigmine, tacrine and other chemical drugs for the treatment of Alzheimer's disease have certain side effects, taking the commercialized traditional Chinese medicine huperzine A (HA) with less side effects as an example, the inhibition of AChE It is also very meaningful to select several traditional Chinese medicines to screen AChE inhibitors for the treatment of Alzheimer's disease.

In recent years, various methods for monitoring AChE activity have been explored, including chemiluminescent, electrochemical, colorimetric Ellman, and fluorometric assays. However, these methods still have unavoidable defects, such as low detection sensitivity, false positive effect, long test time, complicated instruments, etc., which obviously limit the practical application of these methods. At present, the detection method of single signal transmission mode has become increasingly difficult to meet the needs of researchers, and simultaneously outputting chemical changes and visual signals as quantitative signals of instruments has become a favorable direction for the development of nano-biosensors. The colorimetric method has the advantages of simple operation and rapid response, and can be used for on-site naked eye detection.

Nano-biosensors based on enzyme activity regulation have become a hotspot for monitoring target enzymes due to their inherent advantages such as high robustness, sensitivity, and selectivity. As a nanomaterial with enzyme-like activity, nanozyme has attracted more and more attention due to its unique characteristics such as low preparation cost, flexible design, and diverse structures, and has a good prospect for practical application. However, we found that different reactive sites on the catalyst have different catalytic activities, and the catalyst should be designed with more catalytic sites exposed on the edge, which have higher catalytic activity. To this end, the development of two-dimensional (2D) nanomaterials is an effective strategy to enhance the activity of nanozymes, and two-dimensional MOFs have been widely used in catalysis, biosensing, and biomedicine due to their structural advantages of ultrathin thickness and large number of active sites. field. Therefore, the use of 2D MOF nanosheets to construct sensitive colorimetric sensors has attracted extensive research interest.

Contents of the invention

Aiming at the current problems of monitoring AChE activity and its inhibitor HA, the application provides a two-dimensional Cu-CAT nanosheet with enzyme-like activity, which is a two-dimensional (2D) nanomaterial with active sites The advantages of high sensitivity, good specificity, etc., are conducive to the generation of colorimetric signals; at the same time, the application of this nanosheet is also provided, which is used for the colorimetric biosensor for rapid detection of AChE and its inhibitors, and has good detection selectivity for AChE , low detection limit; at the same time, it can detect or screen AChE inhibitors.

Technical scheme of the present invention is as follows:

A two-dimensional Cu-CAT nanosheet with enzyme-like activity, comprising the following raw materials in parts by weight: Cu(OAc) ₂ , sodium dodecyl sulfate, 2,3,6,7,10,11-hexahydroxy Triphenyl hydrate, dextran.

Preferably, it is characterized in that the molar ratio of Cu(OAc) ₂ to 2,3,6,7,10,11-hexahydroxytriphenyl hydrate is 1.5:1.

The preparation method of the above-mentioned two-dimensional Cu-CAT nanosheet with enzyme-like activity comprises the following steps:

(1) Preparation of Cu-CAT: Dissolve Cu(OAc) ₂ and sodium lauryl sulfate in water, add lye; then add 2,3,6,7,10,11-hexahydroxytriphenyl hydrate; React, stand still; collect the precipitate, wash, centrifuge, and dry; obtain Cu-CAT;

(2) Preparation of Cu-CAT nanosheets: Mix Cu-CAT and dextran solution prepared in step (1), react, and centrifuge to remove large pieces of Cu-CAT; centrifuge the supernatant to obtain two-dimensional Cu-CAT NSs .

Further, the reaction conditions of the step (1) are: ultrasonic at 50° C. for 0.5-3 h; the standing conditions are: room temperature for 5-15 h.

Further, the washing process of the step (1) is to wash with ethanol-water solution for more than 3 times at 0-10° C. by ultrasonic.

Further, the reaction condition of the step (2) is ultrasonic for more than 16 hours; the centrifugation condition is: 5000-6000 rpm for 8-15 minutes; the supernatant centrifugation condition is: 13000-15000 rpm for 10-20 min.

Another purpose of the present application is to protect a colorimetric biosensor, including a monochromator, a sample pool, and a detector of a UV-visible spectrophotometer. Add the two-dimensional biosensor prepared by the above preparation method to the sample pool cuvette Cu-CATNSs.

The application of the above colorimetric biosensor is applied to the sensitive detection of AChE and its inhibitors.

The method for detecting AChE and its inhibitors by the above-mentioned colorimetric biosensor comprises the following steps:

S1 Detection of AChE: Mix acetylthiocholine with acetylcholinesterase _AChE and phosphate-buffered saline and incubate; add NaAc-HAc buffer, Cu-CAT NSs, _H2O2 and TMB sequentially; then at room temperature Incubate, record the UV-visible absorption signal at 652nm to detect AChE;

S2 Detection of AChE inhibitors: Mix acetylthiocholine with acetylcholinesterase AChE, AChE inhibitors and phosphate-buffered saline, and incubate; add NaAc-HAc buffer, Cu-CAT NSs, H ₂ O ₂ and TMB; then incubated at room temperature, recorded the UV-visible absorption signal at 652nm to detect AChE.

The enzyme inhibitor of the present invention is a substance that can inhibit the specific enzyme activity related to a certain disease in the organism, so as to obtain curative effect. The nanostructure of two-dimensional Cu-CAT NSs in the present invention is nanosheets, which have ultra-thin thickness and abundant catalytic active sites compared with three-dimensional nanomaterials, thereby improving the efficiency of catalytic oxidation of TMB and further enhancing the performance of colorimetric biosensors. Signal.

Beneficial effects of the present invention

(1) The two-dimensional Cu-CAT NSs prepared by the present invention have the advantages of abundant active sites, good selectivity, and high catalytic efficiency, which are beneficial to generate colorimetric signals.

(2) The novel colorimetric biosensor for rapid detection of AChE prepared by the present invention has high detection sensitivity for AChE and a low detection limit of 0.01 mU mL ^-1 .

(3) Taking huperzine A as an example, the method of the present invention can be used to detect or screen AChE inhibitors, and can also be used to detect whether there are AChE inhibitors with similar effects as huperzine A in Chinese medicine, that is, from traditional Chinese herbal medicines Drugs found in Alzheimer's disease with high sensitivity and specificity.

(4) The present invention shows the development potential of two-dimensional MOFs, and the constructed biosensor provides new insights for the development of new drugs for Alzheimer's disease.

Description of drawings

Figure 1 is a schematic diagram of the process of exfoliating bulk Cu-CAT prepared in Example 1 of the present invention into two-dimensional sheet-like Cu-CAT NSs and its colorimetric biosensing;

Figure 2 is the HRTEM image of Cu-CAT (A) and Cu-CAT NSs (B); (C) the SEM image of Cu-CAT NSs and the corresponding EDS maps of C, Cu and O;

Figure 3 is (A) XRD patterns of Cu-CAT and Cu-CAT NSs; (B) zeta potential of Cu-CAT and Cu-CAT NSs; (C) Cu 2p high-resolution XPS spectra of Cu-CAT NSs and (D) Peak fitting of the LMM spectra of Cu-CAT NSs;

Fig. 4 is (A) Cu-CAT NSs/TMB/H ₂ O ₂ system adding blank, ATCh, AChE and ATCh/AChE UV-Vis absorption spectra; (B) Cu-CAT NSs/TMB/H ₂ O ₂ system UV-Vis absorption spectra and images (inset) of 0.5 mM ATCh and different concentrations of AChE: 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0 mU mL ^- 1 (inset); (C) ΔA The relationship between 652 nm and AChE concentration; inset: AChE linear fitting graph; (D) Inhibition rate (IE%) of different concentrations of HA on AChE. IE% = 100 (A _i -A)/(A ₀ -A)×100%, where A is the absorbance of the system with AChE, A ₀ is the absorbance of the system without AChE and HA, and A _i is the absorbance of the system with HA and AChE The absorbance of the system;

Figure 5 is the inhibition rate (IE%) of different Chinese herbal medicine water extracts (A) and ethanol extracts (B) to acetylcholinesterase; (C) corresponding Chinese herbal medicine images.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

Example 1

A method for preparing a two-dimensional Cu-CAT nanosheet with enzyme-like activity, comprising the following steps:

(1) Add Cu(OAc) ₂ (84.6 mg, 0.465 mmol), sodium dodecyl sulfate (SDS) (50 mg, 0.17 mmol) and 50 mL ultrapure water into a 250 mL Erlenmeyer flask. Then, 50 mL of NaOH solution (50 mg, 1.25 mmol) was added to the above solution, followed by 100 mg (0.31 mmol) of 2,3,6,7,10,11-hexahydroxytriphenyl hydrate (HHTP). Sonicate at 50°C for 0.5 h, and then stand at 25°C for 10 h. The precipitate was collected, washed three times with water and ethanol in an ultrasonic ice bath, each time for 0.5 h, centrifuged to obtain the precipitate, and dried in an oven at 60 °C to obtain a solid powder.

(2) Mix 0.3 g bulk Cu-CAT prepared in step (1) with 4.8 mL dextran solution (2.0 g L ^-1 ), and then sonicate for 16 hours. The resulting solution was centrifuged at 5500 rpm for 10 min to remove bulk Cu-CAT. The supernatant was centrifuged at 13500 rpm for 15 min to obtain two-dimensional Cu-CAT NSs.

The precipitate was dispersed in 2.7 mL of water for further experiments.

Example 2

Add Cu-CAT NSs (0.3mL, 400 μg mL ^-1 ) prepared in Example 1 to a buffer solution with pH=4 (1.5 mL, 200 mM), then add TMB (0.6 mL, 1 mM) and H ₂ O ₂ (0.6 mL, 15 mM). After reacting for 20 min, the UV-Vis absorption spectrum of the system was recorded with a UV-Vis spectrophotometer to measure the peroxidase-like activity of the nanozyme at pH=4.

Example 3

Add Cu-CAT NSs (0.3mL, 400 μg mL ^-1 ) prepared in Example 1 to a buffer solution with pH=7 (1.5 mL, 200 mM), then add TMB (0.6 mL, 1 mM) and H ₂ O ₂ (0.6 mL, 15 mM). After reacting for 20 min, the UV-Vis absorption spectrum of the system was recorded with a UV-Vis spectrophotometer to measure the peroxidase-like activity of the nanozyme at pH=7.

Example 4

Add Cu-CAT NSs (0.3mL, 200 μg mL ^-1 ) prepared in Example 1 to a buffer solution with pH=4 (1.5 mL, 200 mM), then add TMB (0.6 mL, 1 mM) and H ₂ O ₂ (0.6 mL, 15 mM). After 20 min of reaction, the UV-Vis absorption spectrum of the system was recorded with a UV-Vis spectrophotometer, and the peroxidase-like activity of the nanozyme was measured at pH=4.

Example 5

Add Cu-CAT NSs (0.3mL, 600 μg mL ^-1 ) prepared in Example 1 to a buffer solution with pH=4 (1.5 mL, 200 mM), then add TMB (0.6 mL, 1 mM) and H ₂ O ₂ (0.6 mL, 15 mM). After 20 min of reaction, the UV-Vis absorption spectrum of the system was recorded with a UV-Vis spectrophotometer, and the peroxidase-like activity of the nanozyme was measured at pH=4.

As _shown in Figure 1, a new platform was established based on Cu-CAT NSs/TMB/ _H2O2 for the ultrasensitive detection of AChE and HA;

As shown in Figure 2, the structural details of 3D bulk Cu-CAT and 2D Cu-CAT NSs were confirmed by high-resolution transmission electron microscopy (HRTEM). As shown in Figure 2A, the three-dimensional bulk Cu-CAT has irregular shape and large size, and the measured lattice spacing is about 1.72 nm. Whereas the 2D Cu-CAT NSs exhibited a 2D nanosheet morphology (Fig. 2B) with lattice fringes of 1.72 nm, corresponding to the hexagonal crystal system, which could be assigned to the (100) lattice plane. The same 2D and 3D Cu-CAT lattices indicated that the two different morphological MOFs had the same crystal structure; the EDS map analysis of Cu-CAT NSs (Fig. 2C) showed that C, Cu, and O elements were evenly distributed.

As shown in Figure 3, the x-ray diffraction (XRD) spectra of bulk Cu-CAT and Cu-CAT NSs (Figure 3A) data show that 2θ = 4.7, 9.6, 12.8 and 26.8 correspond to (100), (200) , (210) and (002) planes. These results indicated that the crystal structure remained largely unchanged before and after ultrasonic exfoliation, indicating that the dextran-assisted ultrasonic exfoliation method caused less damage to MOFs. The zeta potential of Cu-CAT NSs suspended at -29.27 mV was more negative than that of bulk Cu-CAT at -25.43 mV (Fig. 3B), suggesting that Cu-CAT NSs tended to be more stable and exposed more the active site. The XPS spectrum of Cu 2p (Fig. 3C) shows two doublets of Cu 2p _1/2 and Cu 2p _3/2 . Figure 3D shows the Auger Cu LMM spectrum, three peaks at 570.0 eV (Cu ⁺ ), 568.9 eV (Cu ²⁺ ) and 568.1 eV (Cu ⁰ ) represent different valence states of the Cu LMM spectrum. The existence of Cu ⁺ valence states indicates that Cu ²⁺ ions are partially reduced during the synthesis of bulk Cu-CAT.

As shown in Figure 4A, Cu-CAT NSs catalyzed the oxidation of colorless TMB to blue ox-TMB in the presence of _H2O2 , and formed _a strong absorption peak at 652 nm. With the addition of AChE, the substrate ATCh was hydrolyzed to generate TCh, and the functional group -SH of TCh could bind to the catalytic active site of Cu-CAT NSs to reduce its peroxidase-like activity. Therefore, colorless TMB cannot be oxidized to blue ox-TMB. In contrast, when ATCh or AChE was added alone to the system, the UV-visible absorption spectrum or color of the system remained unchanged (Fig. 4A). These results indicated that pure ATCh and AChE did not affect the absorbance of the sensing system. With the addition of AChE in the range of 0.01-4.0 mU mL ^-1 , the UV-visible absorption intensity at 652 nm of the Cu-CAT NSs/TMB/H ₂ O ₂ /ATh/AChE system gradually decreased (Fig. 4B). After adding AChE, the UV-Vis absorbance quenching efficiency at 652 nm is defined as ΔA ₆₅₂ (ΔA ₆₅₂ = A ₀ - A _i , where A ₀ and A _i represent the absorbance values without adding AChE and adding AChE, respectively), with ΔA652 increased linearly with increasing AChE concentration (Fig. 4C). The regression equation is ΔA ₆₅₂ = 0.08156c +0.01696, and the correlation coefficient (R ² ) is 0.99458. The sensor's limit of detection (LOD) was calculated to be as low as 0.01 mUmL ^-1 (N = 5) based on S/N = 3. In addition, the sensor can be used to screen the inhibitor of Alzheimer's disease, HA, which is an effective drug for the treatment of Alzheimer's disease. In the presence of HA, AChE activity is inhibited, resulting in a decrease in TCh. Therefore, Cu-CAT NSs maintained the corresponding catalytic activity, leading to the recovery of signal from TMB to ox-TMB. In the ATCh/TMB/H ₂ O ₂ system, as the HA concentration increased, the UV-vis absorption intensity at 652 nm increased (Fig. 4D). That is, HA inhibited the activity of AChE and blocked the decomposition of ATCh to generate TCh, thereby restoring the peroxidase-like activity of Cu-CAT NSs. The corrosion inhibition rate (IE%) of AChE increased linearly with the increase of HA concentration in the range of 0 to 200 nM. The linear equation is IE% = (1.14389c + 14.74906)×100%, and the correlation coefficient (R ² ) is 0.98142. The average IC ₅₀ value (half maximum inhibitory concentration) was 30.81 nM. The results show that this method can detect or screen AChE inhibitors.

As shown in Figure 5A, Chinese herbal medicine has become the drug of choice for the treatment of Alzheimer's disease due to its significant curative effect and small side effects. Therefore, the screening of AChE inhibitors from traditional Chinese medicine is of great significance for Alzheimer's disease drug innovation. Eight kinds of Chinese herbal medicines including Chuanxiong (LW), Ophiopogon japonicus (DLT), Acanthopanax (AS), Corydalis (RC), Lycium barbarum (LCM), Cistanche (DC), Radix Paeoniae Rubra (PRR) and Salvia miltiorrhiza (SM) were used as research materials. object. Chinese herbal extracts were prepared with water and ethanol, respectively. Then, using this sensing platform, the water and ethanol extracts were detected by UV-visible absorption spectroscopy and visualization, respectively. As shown in Figure 5A and B, both the water and ethanol extracts of AS and RC had higher inhibitory efficiencies against AChE. This may be due to the protection of dopaminergic neurons in the brain from caspase-induced apoptosis by AS. In addition, RC contains a large amount of cordyrine, an alkaloid that resists AChE. The corresponding herbal image is shown in Figure 5C. These results suggest that the sensor developed in this study is an effective biosensing platform for the detection and screening of acetylcholinesterase and its inhibitors.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (6)

1. the application of colorimetric biosensor on the sensitive detection of AChE and its inhibitor, it is characterized in that, described colorimetric biosensor comprises the monochromator of ultraviolet-visible spectrophotometer, sample pool, detector, in sample Two-dimensional Cu-CAT nanosheets are added to the cell cuvette; The preparation method of described two-dimensional Cu-CAT nano sheet, comprises the following steps: (1) Preparation of Cu-CAT: Dissolve Cu(OAc) ₂ and sodium lauryl sulfate in water, add lye; then add 2,3,6,7,10,11-hexahydroxytriphenyl hydrate; React, stand still; collect the precipitate, wash, centrifuge, and dry; obtain Cu-CAT; (2) Preparation of Cu-CAT nanosheets: Mix Cu-CAT and dextran solution prepared in step (1), react, and centrifuge to remove large pieces of Cu-CAT; centrifuge the supernatant to obtain two-dimensional Cu-CAT nanosheets piece. 2. The application according to claim 1, characterized in that in the step (1), the molar ratio of Cu(OAc) ₂ to 2,3,6,7,10,11-hexahydroxytriphenyl hydrate For: 1.5:1. 3. The application according to claim 1, characterized in that the reaction conditions of the step (1) are: ultrasonic at 50°C for 0.5-3 h; the standing conditions are: 5-15 h at room temperature. 4. The application according to claim 1, characterized in that the washing process in the step (1) is at 0-10°C, ultrasonic washing with ethanol-water solution for more than 3 times. 5. The application according to claim 1, characterized in that, the reaction condition of the step (2) is ultrasonic for more than 16 hours; the centrifugation condition is: centrifugation at 5000-6000 rpm for 8-15 minutes; the supernatant centrifugation condition For: Centrifuge at 13000-15000 rpm for 10-20 min. 6. application according to claim 1, is characterized in that, adopts the method for detecting AChE and inhibitors thereof of colorimetric biosensor described in claim 1, comprises the following steps: S1 Detection of AChE: Mix acetylthiocholine with acetylcholinesterase AChE and phosphate-buffered saline and incubate; add NaAc-HAc buffer _, Cu-CAT nanosheets, _H2O2 and 3,3' in sequence, 5,5'-Tetramethylbenzidine TMB; then incubate at room temperature, record the UV-visible absorption signal at 652nm to detect AChE; S2 Detection of AChE inhibitors: Mix acetylthiocholine with acetylcholinesterase AChE, AChE inhibitors and phosphate-buffered saline, and incubate; add NaAc-HAc buffer, Cu-CAT nanosheets, _{H2O2 in sequence} and 3,3',5,5'-tetramethylbenzidine TMB; then incubated at room temperature, and recorded the UV-visible absorption signal at 652nm to detect AChE.

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